The RAS genes encode small GTPase Ras proteins that are important for controlling cell growth and differentiation. Activating mutations of Ras are associated with approximately 20-30% of all human cancers. All Ras proteins have a C-terminal CaaX motif that is extensively post-translationally modified. These modifications are prerequisite for localization of Ras proteins to the inner leaflet of the plasma membrane where they must be to function properly. It is therefore hypothesized that interfering with the post-translational modification of Ras could be an anti-cancer strategy. The CaaX motif is sequentially modified by the farnesyltransferase (FTase), the Ras converting enzyme (Rce1p), and the isoprenylcysteine carboxylmethyltransferase (ICMT), making them all promising anti-cancer targets. Our long-term goal is to identify agents that interfere with Rce1p. We hypothesize that pharmacological inhibition of Rce1p will lead to Ras mislocalization in vivo. Indeed, we have identified several compounds that inhibit Rce1p in vitro in a species-independent manner (i.e., human, yeast, and trypanosome) and disrupt Ras localization in vivo. This proposal aims to increase the number of similar acting compounds by identifying novel and potent Rce1p-specific inhibitors by high throughput screening (AIM 1). The primary screening of compounds will be performed in collaboration with the Emory HTS facility using a mix and measure fluorescence-based assay and Rce1p as the target enzyme. The specificity of primary hits will be determined using secondary screens to measure effects on two other enzymes, Ste24p and trypsin. Ste24p has a partially overlapping function with Rce1p in that it is required for the post-translational modification of certain CaaX motifs, but it is not specifically required for Ras modification. The expected hit rate using the proposed approach is 0.1% based on preliminary screening of a small compound library (i.e. NCI Diversity Set). The secondary hits will be further characterized by the applicant's laboratory (AIM 2). This analysis will include additional specificity assessments using a distinct reporter and assay system that is based on in vitro production of the yeast isoprenylated a-factor mating pheromone. In vitro approaches will be used to characterize compound in terms of their inhibition kinetics (e.g. KM, KI, vmax), potency (e.g. IC50), and biophysical properties. In vivo cell-based assays will be used to determine toxicity and cell permeability, the latter using a unique GFP-Ras2p localization assay that we have developed. We expect this study to yield several active compounds that will be valuable for understanding the enzymatic nature of the relatively uncharacterized Rce1p and for serving as lead compounds for anti-cancer therapeutics involving targeted inhibition of Rce1p. PUBLIC HEALTH RELEVANCE: The Ras converting enzyme (Rce1p) is an anti-cancer target. High throughput screening for Rce1p- specific inhibitors will be conducted in association with the Emory HTS facility using a direct mix and measure in vitro fluorescence-based primary assay and secondary assays to address target specificity. The identified compounds, representing lead compounds for anti-cancer therapeutics, will be evaluated for their toxicity profile and cell permeability using cell-based assays and will be further assessed for target specificity and kinetic parameters by in vitro methods. [unreadable] [unreadable] [unreadable]